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Effect of the water extracts of Avocado fruit and Cherimoya leaf on
four human cancer cell lines and Vicia faba root tip cells
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Effect of the water extracts of Avocado fruit and
Cherimoya leaf on four human cancer cell lines and Vicia
faba root tip cells
Abstract
The main objective of this study is to determine the effect of the water extract of Persea
americana Mill. (avocado fruit) and Annona cherimolia Mill. (cherimoya leaf) on living cells.
The antiproliferative properties of avocado fruit water extract (AFWE) and cherimoya leaf
water extract (CLWE) were determined using four human cancer cell lines: lung (A549), liver
(HepG-2), colon (HT-29) and breast (MCF-7). Cancer cells were incubated with 100 µg/ml of
AFWE or CLWE for 48 hours, then the cell viability was measured using colorimetric
tetrazolium cleavage test (MTT). Both extracts resulted in more than 90 % mortality in treated
cells. Vicia faba root tip assay was used to determine the effect of AFWE and CLWE on
mitotic index (MI), Micronuclei (MN) formation rate and chromosomal abnormalities. Vicia
faba roots were soaked in 100, 1250, 2500, 5000, 10000, 20000 µg/ml of AFWE or ALWE
for 4 and 24 h. AFWE and CLWE were mitodepressive and resulted in a significant decrease
of MI in a dose dependant manner. CLWE treatment resulted in a decrease in prophase cell
percentage and an increase in MN & chromosomal abnormalities. On contrary, the prophase
cell percentage was linearly increasing with the applied concentration without micronuclei
formation in avocado treatment. Our results strongly indicate that avocado and cherimoya
extracts were highly cytotoxic and mitodepressive on cancer and plant cells, respectively.
Key Words: Annona cherimolia, Persea americana, Micronuclei, mitotic index, Vicia faba
root assay
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1. Introduction
The diverse plant kingdom is gaining global recognition as unique and renewable
resource for the discovery of phytochemicals that may represent pharmacological
active compounds (Am et al., 2013). Phytochemicals are used in many aspects such
as
food supplements (Abrahim et al., 2012; Adebajo et al., 2012), immunity
enhancers (Bayrami &Boskabady, 2013; Benmebarek et al., 2013; Buapool et al.,
2013), wound healers (Chandra et al., 2013), cancer preventive and therapeutic
agents (Bhagat et al., 2012). Cancer prevention through the diet can be considered as
an effective tool to improve public health and reduce the cost for healthcare
(Haniadka et al., 2013). According to Newman et al. (2007), 40% of all anticancer
drugs are developed from natural products while 20% are synthetic derived from
natural ones. However, the detection of an active compound with a biological activity
of interest in about 250000-500000 unexplored plant species is similar to find a
needle in a haystack (Rates, 2001).
The study of natural bioactive compounds in plants has required the
development of bioassay techniques. It is not always possible to test against cancer in
animal models due to the system complexity. In vitro methods allow the screening of
large numbers for cytotoxicity against many types of cancer cell lines and usually
require less test material, time and money (El-Menshawi et al., 2010).
Plant bioassays that measure mitotic cell cycle, micronuclei induction rate and the
frequency of chromosomal aberrations are both time and cost effective. These tests
are helpful in screening the bioactivity of plant extracts at large scale, particularly in
areas with limited funds. They will also eliminate the hazards of using cultured
human cells and experimental animals. Plant bioassays are mainly used in most
ongoing research to determine the genotoxic effect of chemical compounds rather
than testing their pharmaceutical properties.
Root tip meristems of Vicia faba (broad beans) have been used as a pioneer
cytogenetic material for the detection of genotoxicity in many studies (Ma et al.,
2005; Dong &Zhang, 2010). This bioassay was validated by the International
Programme on Chemical Safety (IPCS, WHO) and the United Nations Environment
Program (UNEP). As far as the authors are aware, Vicia faba root tip assay had not
been used for screening natural plant extracts for their anticancer potential.
Persea americana Mill. (avocado) is an economically important tropical tree
belonging to family Lauracea. From phytochemical perspective, extracts of the plant
yield functionalized alkanols known as aliphatic acetogenins (Kawagishi et al.,
2001). Avocado is used
in treating tumor in ethnomedicine and exhibits a
chemoprotective effect on human cells (Paul et al., 2011).
Annona cherimolia Mill. (Cherimoya) is a subtropical tree that belongs to family
Annonaceae. Cherimoya showed
a significant cytotoxic potential in pancreatic,
mammary, prostatic, and kidney cancer cells (Alali et al., 1999). Members of this
family gained more interest due to the presence of the secondary metabolite
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acetogenins. Acetogenins can be used as antiparasitic, antimicrobial, antimicotic,
immunosuppressive, cytotoxic and antitumorous agents (Garcia-Aguirre et al., 2008).
Avocado and cherimoya are known to posses stronger cytotoxic effect in
tumorous than in normal cells. Some studies showed that plant and animal cells
exhibited similar responses towards treatments with bioactive compounds (Konuk et
al., 2007). To determine of this is the case, we tested the cytotoxic effect of avocado
fruit water extract (AFWE) and cherimoya leaf water extract (CLWE) on four human
cancer cells and meristematic plant cells. The bioactivity of the water extracts of
avocado and cherimoya had never been tested on human cancer cell lines or plant
cells else where. Therefore, this study will be very helpful in highlighting the
activities of both extracts and also to compare between the efficiency of the two
bioassays in determining their bioactivity. For this aim, the anticancer properties of
ALWE and CLWE were determined using four human cancer cell lines: A549
(human lung carcinoma), HePG2 (human liver carcinoma), HT29 (human colon
carcinoma) and MCF-7 (human breast carcinoma). The genotoxic effects of the two
extracts were determined by measuring the mitotic index (MI), micronuclei (MN)
formation rate and chromosomal abnormalities in Vicia faba root tip cells.
2. Materials and Methods
2.1 Plant materials
Fresh samples of Persea americana fruit and Annona cherimoli leaves were obtained
from El Orman Botanical Garden (Giza, Egypt). The plants were identified by the
herbarium officer of El Orman Garden: Mrs Teriz Labib. Voucher specimens were
kept in the herbarium, Botany Department, Faculty of Science, Ain Shams
University, under the name of Noha-Dina (2009). Leaves were collected at
approximately similar age (the third fully expanded leaves from the top of each
branch) and fully ripened dry fruits of P. americana were used. Seeds of Vicia faba
(cultivar: Windsor white) were kindly provided by the Agriculture Research Centre,
Ministry of Agriculture, Giza, Egypt and was used as a reference model in the root
tip bioassay.
2.2 Preparation of extracts
Water extracts were prepared by dissolving 2 grams of fresh material in 100 ml of 90
ºC distilled water, incubated at the same temperature for 15 min, cooled, filtered and
used as a stock (2000000 µg/ml). Water extracts were prepared and used the same
day of collection to avoid any change in its activity.
2.3 Cell culture
A549 (human lung carcinoma), HePG2 (human liver carcinoma), HT29 (human
colon carcinoma) and MCF-7 (human breast carcinoma) were obtained from
Karolinska Institutet , Stockholm, Sweden. All cells were maintained in RPMI 1640
(Lonza Biowahittkar) medium except for MCF-7 cells which were maintained in
DMEM medium (Lonza Biowahittkar). All the media were supplemented with 1 %
antibiotic-antimycotic mixture (10.000 U/ml Potassium Penicillin, 10.000 µg/ml
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Streptomycin Sulfate and 25 µg/ml Amphotericin B and 1 % L-glutamine. All
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in 1N analar HCl at 58-60 C for 10 min (Qian, 2004). Root tips- 3 mm long- were
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concentration. Preparations were mounted in Canada balsam and placed at 45 C in
antibiotics and L- glutamine were purchased from Biowest, France.
2.4 MTT viability Assay
The viability of cancer cell lines was determined after the exposure to water extracts
for 48 h using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay as described in Mosmann (1983).
2.5 Root tip preparations and treatment
Vicia faba dry seeds were rinsed and then soaked in distilled water for 24 h.
Germinating seedlings were kept at 25 ºC on moist gauze until their primary roots
were 1-2 cm in length (Cotelle et al., 1999). Root tips were soaked for 4 or 24 h in
different concentrations of water extracts (100, 1250, 2500, 5000, 10000, 20000
µg/ml). Distilled water was used as negative control. All treatments were done in
triplicates at temperature of 25 °C ± 2.
2.6 Cytological study and slide preparation
Root-tips were cut directly into Carnoy’s solution [absolute alcohol: glacial acetic
acid (3:1)] and incubated at room temperature for 24 h. Roots are then hydrolyzed
squashed and stained according to Darlington and La-Cour, (1976) Leuco-basic
Fuchsin technique. Light green dye (0.3 %) was used for background staining of the
cytoplasm. Root tips were squashed in 45 % acetic acid. Dehydration was done using
ascending series of ethyl alcohols; 30 %, 50 %, 70 %, 96 %, absolute alcohol;
absolute alcohol: xylene (1:1) and xylene, keeping root tips for 5 min in each
the oven (until completely dry). Root tips were examined for micronuclei frequencies
and other abnormalities at 4000x magnification using Leica light microscope. The
photomicrographs were taken from the prepared slides using digital camera (Sony,
8Mp).
2.7 Mitotic indices (MI) were calculated using the following formula:
Mitotic
index
(MI)
=
Number
of
dividing
cells
X
100
Total cells examined
2.8 Statistical analysis
Data shown are the means and standard errors of three or more independent
experiments. Statistical comparisons between groups were made by Student’s t-test
using Microsoft excel program, and a P-value<0.05 considered to be statistically
significant.
3. Results
3.1 Cytotoxic effect of plant extracts on cancer cell lines
Avocado fruit water extract (AFWE) exhibited strong cytotoxic effect against all
cancer cell lines used in this study (Table 1).
The extract resulted in lethal
percentages of 93.3 %, 98.3 %, 97.8 % and 91.7% in A-549 HepG-2 HT-29 and
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MCF-7 human cancer cell lines, respectively. The cytotoxic effect of avocado extract
was more pronounced as compared to that of cherimoya leaf water extract (CLWE)
in HepG-2 and HT-29 cancer cell lines. The cytotoxic concentrations of the two
extracts were also tested over a range of dilutions on the four cancer cell lines to
determine their LC50 (Table 2 and Fig.1).
Both P. americana and A. cherimolia
water extracts showed LC50 value less than 30 µg/ml in HepG-2 cell lines with LC50
=13.3 & 10 µg/ml and HT-29 cell line with LC50 = 13.3 & 16 µg/ml, respectively.
3.2 The effect of plant extracts on mitotic indices
As shown in Table (3), we observed a decrease in the mitotic indices of root tip cells
of Vicia faba in a dose-dependant manner in all plant extracts used. Differences in
mitotic indices between the treated plants and the untreated control were significant
at the highest concentrations of 10000 and 20000 ppm, applied for 4 h, for all
treatments applied. In 24h treatments, the mitotic indices were significantly reduced
as compared with control at concentrations of 5000, 10000 and 20000 ppm.
3.3 Micronuclei formation rate of Vicia faba root tip cells
The water extract of A. cherimolia was able to induce micronuclei formation (Table
4). In plants treated with higher concentrations (10000, 20000 ppm) of A. cherimolia
water extract, we observed more than one micronucleus per cell (Fig. 2a) with the
formation of nuclear bud (Fig. 2b). Interestingly, no micronuclei were detected in the
root cells of Vicia faba plants treated with all the used concentrations of P.
americana either for 4 or 24 h (Table 4).
3.4 Chromosome aberration rate of Vicia faba root tip cells
Root tip cells treated with the plant extract of P. americana exhibited the least
chromosomal abnormalities as compared with the corresponding control. This result
applies for both treatment duration used (4 or 24h). The most common abnormalities
were stickiness (Fig. 3a) in plants treated with the water extract of P. americana fruit
at relatively high concentrations. On the other hand, Chromosome laggards (Fig. 3b)
and disturbed metaphase chromosomes (Fig. 3c), in addition to stickiness, are the
most common abnormalities detected in plants treated with the leaf water extract of
A. cherimolia.
3.5 Percentage of different mitotic phases
As shown in Fig. 4, the percentage of prophase cells were decreasing in a dose
dependant manner in plants treated with P. americana or A. cherimolia water extracts
for 4 h. The same is true for A. cherimolia treated plants for 24 h. On the other hand,
the percentage of prophase cells was increasing linearly with the concentration
applied in plants treated with P. americana for 24 h (Fig. 4b). The metaphase and
ana-telophase percentages were increasing as well in plants treated with CLWE for
24 h, while they decreased in plants treated with AFWE for 24 h.
4. Discussion
Many studies, including our own, showed that phytochemicals extracted from Persea
americana (avocado) and Annona cherimolia (cherimoya) can selectively induce cell
cycle arrest, inhibit growth and enhance apoptosis in some cancer cell lines other
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than those used here (Ding et al., 2007; Garcia-Aguirre et al., 2008; Ambrosio et al.,
2011). For instance, in vitro studies had shown that the acetone extract of avocado
fruit inhibited the growth of prostate cancer cells
(Lu et al., 2005) while its
chloroform extract inhibited the growth of oral cancer cells (Ding et al., 2009).
Fractions from the ethanol extract of cherimoya inhibited the growth of human colon
cancer and increased the micronuclei formation in mice erythrocytes (Garcia-Aguirre
et al., 2008). However, the effect of avocado fruit water extract (AFWE) and
cherimoya leaf water extract (ALWE) have not been reported before either on cancer
or plant cells, as far as the authors are aware of. This prompted us to test the
properties of AFWE and ALWE in order to mimic the natural way of their
consumption by humans. This was carried out using two bio-screeing approaches: 1cancer cell MTT and 2- Vicia faba root tip - bioassays.
In order to measure their cytotoxic properties, Four cancer cell lines [lung (A549),
liver (HepG-2), colon (HT-29) and breast (MCF-7)] were incubated with 100 µg/ml
of AFWE or CLWE for 48 h.
The Viability of cancer cells was measured using
MTT assay. Both extracts resulted in more than 90% growth inhibition in all cancer
cell lines as shown in Table 1. This indicates that AFWE and CLWE posses the
ability to arrest the growth of cancer cells used in this study. To determine the
concentration at which AFWE and ALWE can inhibit 50% of cancer cell growth, we
treated all cell lines with series of diluted concentrations and measured cell viability
using MTT assay. LC50 values are shown in Table 2 for both extracts. Avocado and
cherimoya water extract exhibited low LC50 = 13.3 & 10 µg/ml in HepG-2 and LC50
= 22 & 16 µg/ml in HT-29 cell lines, respectively. Therefore, avocado and cherimoya
represent very promising sources for anticancer drugs at least for liver and colon
cancers.
In the second approach, we used plant system to determine the effect of AFWE and
CLWE on mitotic index (MI), micronuclei (MN) formation rate and chromosomal
abnormalities. MI , MN and chromosomal abnormalities are well recognized markers
to measure the mitodepressive and genotoxic properties of a test substance using
plant cells. To date, there are no published data assessing the effect of avocado or
cherimoya on Vicia faba root tip cells. Roots of Vicia faba plants were treated with
100, 1250, 2500, 5000, 10000, 20000 µg/ml of AFWE or ALWE for 4 and 24 h. We
have noticed a decrease in mitotic index values within root-tips treated with AFWE
or ALWE, as compared with the untreated control (Table 3). This decrease was more
pronounced in case of AFWE treatments (Table 3). This indicates that both extracts
are mitodepressive.
Mitodepressive effect had been assumed to result from the
inhibition of cells access to mitosis (Badr &Ibrahim, 1987) which is likely attained
by preventing DNA biosynthesis or / and microtubule and chromatin organization
(Yüzbaşıoğlu et al., 2003). This will lead into a slower progression of cells from S
(DNA synthesis) phase to M (mitosis) phase of the cell cycle (Blakemore et al.,
2013).
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The percentages of different mitotic phases (Prophase, metaphase, and ana-telophase)
were decreasing in a dose dependant manner in AFWE (4 h) and CLWE (4 and 24 h)
treated plants (Figs. 1-a, c & d). When plants treated with AFWE for 24 h, the
prophase cell percentage was progressively increasing in a dose dependant manner
(Fig. 1b). Thus, it can be assumed that AFWE may have arrested cell division via
interfering with DNA biosynthesis rather than affecting other stages in cell cycle.
The induction of micronuclei (MN) had been used in many studies as an indicator of
genotoxicity (Cavas &Ergene-Gozukara, 2005). In this work, the genotoxic effect of
AFWE and CLWE was determined using the frequency of micronuclei (MN)
formation. This will give us an idea about the safety of using these extracts in cancer
therapies in future applications. We observed that the decrease in the mitotic index
was associated with a significant increase in micronuclei (MN) formation rate in
ALWE treated plants at all concentrations used (Table 4). We also observed the
formation of two micronuclei per cell (Fig. 2 a) and nuclear bud (Fig. 2 b) at higher
concentrations of CLWE (10000 and 20000 µg/ml). Interestingly, no micronuclei
were detected in plants treated with all concentrations of AFWE.
Chromosomal stickiness was the only abnormality observed at a significant level in
AFWE treated plants. This gives an indication that the mitodepressive effect of
avocado is more potent to an extent that it may had prohibited further cell division.
On contrary, the clastogenic effect of cherimoya included chromosomal stickiness,
bridges, laggards and disturbed metaphase. In this connection,
the presence of
micronuclei is commonly associated with structural and numerical chromosomal
aberrations induced by clastogenic agents that cause chromosomal breaks and
aneugenic agents that disturb microtubule (Bellini et al., 2006; Dufour et al., 2006;
Benfenati et al., 2009). This result suggests that both extracts interfered with the cell
cycle. It can also be assumed that CLWE may be affecting chromatin and
microtubule organization as indicated by micronuclei formation and chromosomal
breaks. The mode of action of AFWE and CLWE as Mitodepressive agents needs
further investigation.
5. Conclusion
This study highlighted the value of avocado fruit and cherimoya leaves to be used as
promising sources for anticancer drugs. Both extracts showed potent cytotoxic
activity toward cancer cells. However, avocado should be given more attention in this
respect due to less chromosomal abnormalities associated with its treatment as
compared to cherimoya.
This study can also be used to compare between the efficiency of MTT cancer cell
viability and root tip assay in determining the cytotoxic properties of a plant extract
toward cancer cells. Our results indicated that both bioassays can lead into the same
conclusion. Thus, Vicia faba root tip assay can be used as an initial screening step
particularly when large number of extracts is involved. The initial screening can then
be followed by extra analysis for the promising extracts to determine the effective
dose, selectivity, ……etc using animal models and human cell lines. When it comes
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to plant bioassay, we found that reduction in mitotic index may indicate the cytotoxic
effect of a plant extract on cancer cells. However, MN frequency and chromosomal
abnormalities should be considered to validate the bio safety of the extract under
investigation.
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2
3
Table 1. In
vitro cytotoxic activity of crude
methanolic extracts tested against human
carcinoma cell lines (A549, HepG-2, HT-29 & MCF-7) after 24 hours.
Plant name
Mean A-549
Mean HepG-2
Mean HT-29
Mean MCF-7
Persea americana fruit
93.3
98.3
97.8
91.7
+ve control (Annona Cherimolia)
95
94
93
94
DMSO
0
0
0
0
-Ve control
0
0
0
0
Blank
-
-
-
-
4
5
6
Table 2: LC50 values related to MTT assay of the water extract of avocado fruit and cherimoya
leaf on human cancer cell lines. Values are in µg/ml.
Lethal concentration (LC50)
Cell line
Avocado
fruit
water
Cherimoya Leaf Water
extract (AFWE)
extract (ALWE)
A-549
35.4
9.8
HepG-2
13.3
10
HT-29
22
9.7
MCF-7
54.5
6.5
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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1
Table 3 The effect of plant extracts on the mitotic index of Vicia faba L.
Plant extract
iT
em
Persea americana
fe me
cnoC. µg/ml
nu
Annona cherimolia
IM ( X ±
fe me nu Cmrr
IM ( X ± SE)
Cmrr n melmc
SE)
n melmc
100
3360
2.98 ± 0.57
3078
3.44 ± 0.55
1250
4200
2.86 ± 0.39
3850
3.17 ± 0.43
2500
4117
2.77 ± 0.49
4077
2.87 ± 0.38
5000
3640
1.92 ± 0.68
4428
2.14 ± 0.55
10000
4200
1.50 ± 0.61*
3630
1.63 ± 0.81*
20000
4944
1.03
3376
1.48 ± 0.73*
4 hours
Control
±
0.14**
Control
100
3516
8.08 ± 0.68
3033
8.08 ± 0.70
1250
3450
7.68 ± 0.90
4015
6.95 ± 0.55
2500
4322
6.94 ± 0.77
4115
6.32 ± 0.86
5000
4200
4.29 ± 0.33
3321
4.67 ± 0.33**
±
3130
3.80 ± 0.56**
2.11 ± 0.24
3900
3.08 ± 0.41**
**
10000
4023
3.11
24 hours
0.38**
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20000
3697
**
Note: * P<0.05, **P,0.001 as compared to the untreated control plants.
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1
24 hours
4 hours
Time
Table 4 Effect of plant extracts on micronucleus formation rate in Vicia faba root tip cells.
Treatment
Micronuclei formation rate (%)
a ac PePa aesPeP
aeeoePesh aeaoneP
Control
---
---
100
---
1.68
1250
---
1.86
2500
---
2.12
5000
---
2.83
10000
---
3.23
20000
---
3.40
Control
---
---
100
---
1.79
1250
---
1.93
2500
---
2.03
5000
---
2.45
10000
---
3.37
20000
---
3.80
2
3
4
5
6
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1
2
Table 5 The percentage of normal and abnormal mitosis in plant cells treated with the water
extracts
Plant extract
of
P.
americana
and
A.
cherimolia
Persea americana
at
different
concentrations.
Annona Cherimolia
4 hours
Time
neeor
cnoC.
Normal
µg/ml
)%(
Anaphase
Impoteo
(%)
Control
26.40
23.58
100
25.00
1250
Abn. Mitosis
Metaphase
Anaphase
Abn. Mitosis
(%)
(%)
(%)
1.88 ± 0.61
26.40
23.58
1.88 ± 0.61
25.00
3.00 ± 0.92
29.25
20.75
3.92 ± 0.60
24.17
23.33
4.17 ± 0.69
27.05
25.41
5.74 ± 1.85
2500
26.32
26.32
4.39 ± 1.18
29.06
28.20
6.84 ± 2.43
5000
28.57
25.71
5.71 ± 1.95
36.84
25.26
8.40 ± 2.47*
10000
30.16
23.80
39.00
23.73
16.95
20000
33.33
23.53
(%)
7.94 ± 2.60*
13.73
±
1.32**
40.00
26.00
1.35**
26.19
±
1.4**
cnopenr
24.30
27.50
1.60 ± 0.57
24.30
27.50
1.60 ± 0.18
100
24.65
26.76
2.46 ± 0.46
22.45
27.76
1.63 ± 0.143
1250
24.15
26.42
3.77 ± 1.00
28.67
26.88
7.17± 3.03
2500
25.67
22.67
33.85
24.62
10.40
5000
10000
20.56
20.00
23.89
21.60
5.00 ± 1.91
45.16
7.78 ± 2.66
16.00
20000
20.51
21.79
24.36
0.55**
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±
3.38*
17.42
23.23±0.83*
*
±
41.18
18.49
1.41**
enfe 42
±
36.13
±
1.2**
±
47.50
19.17
45.83
1.45**
±
1
Figures
LC50 values of avocado and cherim oya on the cancer cell lines
used in this study
Concentration in ug/ml
60
Avocado
50
Cherimoya
40
30
20
10
0
A-549
HepG-2
HT-29
MCF-7
Cancer cell line
2
3
4
5
Figure 1 : LC50 of the water extract of Persea americana fruit and Annona cherimolia leaves in relation to the
four cancer cells lines
[Insert running title of <72 characters]
a
1
2
3
4
5
6
7
8
9
b
Figure 2. Effect of A. cherimolia water extract on micronuclei formation rate. Micronuclei were linearly
increasing in a dose dependant manner (a) Two micronuclei per cells and (c) nuclear bud were observed at
higher concnetrations.
a
10
b
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
c
Figure 3. Chromsomal abnormalities induced upon treatments with the water extracts under study.
Representative micrograph pictures showing the most common abnormalities. (a) Chromosomal stickiness is
commonly observed in plants treated with the highest concentrations of
P. americana and most of the
concentrations of A. cherimolia (b) Disturbed metaphase and (c) chromosomal laggards were only observed in
plants treated with the water extract of A. cherimolia leaves.
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)
50
70
a
40
Proph
as
Metap e
ha
Anaph se
ase
30
Mitotic phases (%)
Mito
tic ph
ases
(%
60
60
Prophase
Metaphase
Anaphase
b
50
40
20
30
10
20
0
Contro
l
10
100
1250
0
2500
Control
5000
centr
at
Mitotic phases (%)
ion O
1250
2500
5000
10000
20000
2000
0
f P. am
erican
60
c
a in
ppm
Prophase
Metaphase
Anaphase
50
60
d
Prophase
Metaphase
Anaphase
50
40
40
30
30
20
20
10
10
0
0
Control
2
3
4
5
100
conc. Of P. americana in ppm
Con
Mitotic phases (%)
1
1000
0
100
1250
2500
5000
10000
20000
Concentration of Annona in ppm
Control
100
1250
2500
5000
10000
20000
Concentration of Annona in ppm
Figure 4. Mean values of different mitotic phases of treated and untreated V. faba root tip cells. Effect of water
extract of P. americana (a): for 4 and (b): 24 h. Effect of the water extract of A. cherimolia leaves (c): for 4
and (d): 24 h.
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